Title of Invention: IMP production method using the new polypeptide, and this

Art

[1]

The present application relates to a novel protein variant, microorganisms, 5'-inosinate production method and the method of increasing the discharge 5'-inosinate using the same comprising the same having a 5'-inosinate emptying.

[2]

BACKGROUND

[3]

One of the nucleic acid-based materials 5'-inosinate (5'-inosine monophosphate; hereinafter IMP) has been used in various fields as an intermediate material of the nucleic acid metabolic pathway, food, drugs and other medical use and the like. In particular, 5'-guanylate; a material that is widely used as a seasoning for foods or food additives, with (5'-guanine monophosphate below GMP). IMP is becoming a known, but that the beef flavor by itself, mono sodium glutamate (MSG) known to enhance the flavor of the positive immature nucleic acid-based seasoning.

[4]

A method of producing the IMP is a method of decomposing ribonucleic acid extracted from yeast cells by the enzymatic method (Japanese Patent Publication No. 1614/1957 call) with a chemical phosphorylation of inosine produced by fermentation (Agri. Biol. Chem ., 36, 1511 there is, and so on) and a method for culturing a microorganism producing IMP and recovering the IMP directly in the culture medium or the like. How are currently the most widely used of these methods is a method using a microorganism capable of producing direct IMP.

[5]

On the other hand, in the natural state, enzymes like substrate specificity of the activity, stability, and optical isomers that are required for the industrial utilization is always different to improve the suitable enzyme in use for the purpose does not indicate optimum properties through such variations in the amino acid sequence attempts have been made. Among them, examples of rational design (rational design) and partial mutation (site-directed mutagenesis) methods of enzyme applied to improve the enzyme function. However, in many cases, the lack of information about the structure of the target enzyme or structure-correlation function It has the disadvantage that it can be effectively applied to relationships unclear. There it is also reported methods to try to improve the orientation of the enzyme through evolution for screening the enzymes for the desired trait from the mutated enzyme libraries built from a random mutation of the gene (directed evolution) ways to improve the activity.

[6]

Detailed Description of the Invention

SUMMARY

[7]

In order to produce a high yield of the IMP to a method of directly producing IMP by fermentation of microorganisms of the IMP and the exhaust it must be made smoothly. In order to achieve this purpose the present inventors have completed the present application by the excavation protein variants with the result sought example, IMP emptying in order to identify the proteins involved neunge IMP emissions and increases the IMP production.

[8]

Problem solving means

[9]

One object of the present application is to provide a mutant protein for discharging the 5'-inosinate.

[10]

Another object of the present application is to provide a polynucleotide encoding the protein variants of the present application.

[11]

It is another object of the present application is to provide a vector comprising a polynucleotide of the present application.

[12]

It is another object of the present application is to provide a microorganism producing 5'-inosinate which comprises a vector of the protein variants and the application of the present application.

[13]

It is another object of the present application is to provide a 5'-inosinate manufacturing method comprising the step of culturing a microorganism in a culture medium of the present application.

[14]

It is another object of the present application is to provide a 5'-inosinate exhaust protein variant the Corynebacterium genus Solarium method of increasing the discharge, 5'-inosinate which comprises enhanced in the microorganism of the present application.

[15]

Effects of the Invention

[16]

When culturing a microorganism of the genus Corynebacterium by using the protein variant to discharge the IMP of the present application to produce 5'-inosinate, 5'-inosinate is possible to produce a high yield.

[17]

Best Mode for Carrying Out the Invention

[18]

If it described in detail below. On the other hand, each of the descriptions and embodiments disclosed in this application may be applied to other embodiments and description of each. That is, any combination of various elements disclosed in the present application within the scope of the present application. In addition, it is impossible to see that the scope of the present application limited by the specific description technology.

[19]

[20]

One aspect of the present application for achieving the above object is to provide a mutant protein for discharging the 5'-inosinate.

[21]

[22]

Term in this application, the terms "protein for discharging the 5'-inosinate" is 5'-inosinate (5'-inosine monophosphate; IMP) means a protein that participates in discharged outside the cell. For purposes of this application the terms may be used interchangeably as the IMP protein having a discharge capacity, discharge IMP protein, the protein having a 5'-inosinate emptying, 5'-inosinate exhaust protein. More specifically, the protein can be represented by ImpE, not intended to be more specifically, but may represent ImpE1 or ImpE2, limited. In addition, the proteins may be derived from genus Corynebacterium, in particular, but may be a Corynebacterium origin varnish letting stay, but is not limited thereto.

[23]

The protein may for example be one consisting of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, but includes no sequences that have the same activity as the protein is limited, the sequence information, etc. One of ordinary skill in the art GenBank of the known database NCBI It can be obtained. In addition, the protein is SEQ ID NO: 1 or SEQ ID NO: 2 in amino acid sequence or the at least 80%, of the amino acid sequence having 90%, 95%, 96%, 97%, 98%, or 99% homology or identity which may be the protein. In addition, having such homology or identity if the amino acid sequence shown efficacy corresponding to the protein, with some sequences do proteins having deleted, modified, substituted or added in the amino acid sequence can be used as a protein of the present application will be apparent.

[24]

That is, even if in the present application are described as "protein consisting of a specific SEQ ID NO: Amino Acid Sequence", a protein comprising the amino acid sequence set forth in a particular SEQ ID number, or a protein with the same or equivalent activity consisting of the amino acid sequence of the SEQ ID NO: If the case has, with some sequences do protein having a deletion, modification, substitution, conservative substitutions or added in the amino acid sequence may be used in the present application will be apparent. For example, the variant protein with the same or a case having a corresponding activity, if the amino acid sequences before and after additional sequence that does not change the function of the protein, the mutations may occur naturally, their potential mutations (silent mutation) or retention not intended to exclude the substitution, it is apparent that within the scope of the present application, even if having more such sequences or mutations.

[25]

[26]

The term in the present application, "homology (homology) 'or' identity (identity)" means the degree to each other relating to the two given amino acid sequence or nucleotide sequence and can be expressed as a percentage.

[27]

Terms homology and identity can often be used interchangeably.

[28]

A conserved (conserved) polynucleotide or sequence homology or identity of polypeptides can be used with the default gap penalties established by the program that is determined by the standard arrangement algorithm used. In practice, it has a homology or (homologous) or the same (identical) sequences are generally sequences in whole or in full in the middle or high stringent conditions (stringent conditions) - at least 50% of the length, 60%, 70%, 80% or it may be a hybrid by at least 90%. Hybridization is also considered a polynucleotide containing a degenerate codons instead of the codons in the polynucleotide.

[29]

Whether any two polynucleotide or polypeptide sequence of the has the homology, similarity or identity include, for example, Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: by using the default parameters as in 2444 may be determined using known computer algorithms such as the "FASTA" program. Alternatively, only the needles of the EMBOSS package program, as is done in (EMBOSS: 276-277: The European Molecular Biology Open Software Suite, Rice et al, 2000, Trends Genet 16..) (Version 5.0.0 or later) needle-only-flavor (Needleman-Wunsch) algorithm (.. Needleman and Wunsch, 1970, J. Mol Biol 48: 443-453) can be determined is used. (GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215] : 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED,.] Academic Press, San Diego, 1994, and [CARILLO ETA /.] (1988) SIAM J Applied Math 48: 1073 contains) for example, you can use the BLAST, ClustalW or the National Center for Biotechnology information database to determine the homology, similarity or identity.

[30]

Polynucleotides or polypeptides of homology, similarity or identity, e.g., Smith and Waterman, Adv. Appl. Math (1981) 2: 482, as is known in, e.g., Needleman et al. (1970), J Mol Biol.48: can be determined by using the GAP computer program, such as 443 compares the sequence information. In summary, GAP program defines a value obtained by dividing the total number of symbols in the shorter of the two sequences from the number, the arrangement similar to sign (i.e., nucleotides or amino acids). The default parameters for the GAP program include: (1) one binary comparison matrix (1 and ratio to the identity-by containing a value of 0 for identity) and Schwartz and Dayhoff, eds, Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. As disclosed by 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: the weighted comparison matrix of 6745 (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) addition of 0.10 penalty for each symbol in each gap, and the penalty of 3.0 for each gap (or gap opening penalty 10, gap extension penalty 0.5); And (3) may include no penalty for end gaps. Thus, as used herein, the term "homology" or "identity" represents the relevance (relevance) between the sequences. Specifically, the mutant protein having a 5'-inosinate emptying of the present application in 164 amino acid SEQ ID NO: 1, SEQ ID NO: consisting of from 1 to 222 amino acid, the second amino acid, and the 64th amino acid in SEQ ID NO: 2 in Sequence Number 2 one or more amino acids selected from the group may be substituted with a different amino acid, but is not limited thereto.

[31]

For example, the mutant protein having the 5'-inosinate is emptying, or the 164th amino acid in SEQ ID NO: 1 replaced by lysine, arginine, asparagine, glycine, threonine or proline, the second amino acid from the SEQ ID NO: 2 this can be isoleucine, phenylalanine, methionine, glutamic acid, substituted with histidine, or asparagine, or or the 64th amino acid is aspartate, the protein variants replaced with glutamic acid, asparagine, cysteine, isoleucine, or phenylalanine in the sequence No. 2 but are not limited to, .

[32]

Specific example, the amino acid sequence variant protein having the 5'-inosinate emptying is encoded by the polynucleotide of SEQ ID NO: 141, 142, 145, 147, 149 or 151 amino acid sequence, or SEQ ID NO: 153 or 154 made of a or it may be this way at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% of proteins having an amino acid sequence having a homology. Further, if the protein having such a homology with the amino acid sequence shown efficacy corresponding to the protein, with some sequences do proteins having deleted, modified, substituted or added in the amino acid sequence can be used as a protein of the present application, it is obvious .

[33]

[34]

Another aspect of the present application is to provide a vector containing a polynucleotide, or a polynucleotide encoding the protein variants.

[35]

Term in this application, the terms "polynucleotide" is a nucleotide units (monomer) is a polymer (polymer) DNA or RNA strands over a predetermined length to the nucleotide long resulted in a chain shape by a covalent bond, specifically to the variant polypeptide than It means a polynucleotide fragment encoding.

[36]

A polynucleotide of the present application is codon-axis made by the degeneracy (codon degeneracy) of the amino acid sequence encoded by the polynucleotides of the SEQ ID NO: 141, 142, 145, 147, 149 or 151 amino acid sequence, or SEQ ID NO: 153 or 154 of the also it may include a polynucleotide that may be translated to a protein of the protein or the homology is apparent. For example, a polynucleotide of the present application is SEQ ID NO: 143, 144, 146, 148, 150, 152, may be a polynucleotide having a nucleotide sequence of 153 or 154, in particular 143, 144, 146, 148, 150 than , 152, 153 or may be a polynucleotide consisting of a nucleotide sequence of 154. Also, which may be prepared from a known gene sequence probe, e.g., by Hydride Chemistry under the complementary sequences and stringent conditions to all or a portion of the nucleotide sequence of SEQ ID NO: 141, 142, 145, 147, 149 or 151 If the amino acid sequence, or the polynucleotide sequence of SEQ ID nO: protein having the activity of a protein consisting of the amino acid sequence encoded by the polynucleotide of encoding 153 or 154 (i.e., encryption) may be included without limitation.

[37]

Refers to conditions that permit the specific hybridization between the "stringent condition" is a polynucleotide. These conditions are described in detail in the literature (e.g., J. Sambrook et al., Above). For example, the high homology gene each other, more than 40%, specifically, by at least 90%, more specifically at least 95%, more specifically 97% or more, and particularly particularly genes having at least 99% homologous between the hybridization, more than condition homology do not hybridize with each other low gene, or the conventional 60 ℃ washing conditions of Southern hybridization, 1XSSC, 0.1% SDS, specifically 60 ℃, 0.1XSSC, 0.1% SDS specifically, as in the salt concentration and temperature corresponding to 68 ℃, 0.1XSSC, 0.1% SDS, 1 time, and specifically may be exemplified by conditions under which washing twice or three times.

[38]

Hybridization Although the mismatch (mismatch) between the base be possible depending on the stringency of hybridization, though, requires that the two nucleic acids having a complementary sequence. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize to each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, this application can also, as well as similar to the nucleic acid sequence substantially complementary to an isolated nucleic acid fragment comprising the sequence throughout.

[39]

Specifically, a polynucleotide having a homology can be detected using hybridization conditions comprising a hybridization step at Tm value of 55 ℃ using the above-described conditions. Further, the Tm value can be 60 ℃, 63 ℃ or 65 ℃. However, it is not limited to, it can be properly adjusted by those skilled in the art according to the purpose.

[40]

Appropriate stringency for hybridizing polynucleotide is dependent on the degree of complementarity and the length of the polynucleotide and variables are well known in the art (see Sambrook et al., Supra, 9.50-9.51, 11.7-11.8).

[41]

[42]

The term "vector" as used in this application refers to a DNA preparation containing the nucleotide sequence of the polynucleotide encoding the desired protein operably linked to suitable control sequences so as to express the desired protein in a suitable host. The control sequences may include any operator sequence, sequences that control the termination of the sequence, and a transcription and translation encoding a suitable mRNA ribosome-binding site for regulating the promoter, such that transcription can initiate transcription. Vector may then be transformed into a suitable host cell, replicate independently of the host genome, or functions, may be integrated into the genome itself.

[43]

Vector used in the present application as long as it can replicate in a host cell is not particularly limited, it is possible to use any vector known in the art. Examples of the normal vector to be used may be a naturally occurring or recombinant plasmid of the state, cosmid, virus and bacteriophage. For example, the phage vector or course as mid vector may be used. PWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, etc. Charon4A, and Charon21A, pBR series, pUC system, pBluescriptII system as plasmid vector It may be used based pGEM, pTZ-based, such as pCL and pET-based system. Specifically, it may be used pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector or the like.

[44]

With a polynucleotide via a vector for cell insertion mutations within the chromosome of a polynucleotide encoding a protein of interest in the chromosome in one example it can be replaced. Insertion into the chromosome of the above polynucleotides is any method known in the art, for example, but may be made by homologous recombination, but is not limited to this. It may further comprise a selectable marker (selection marker) to determine the chromosomal insertion. Selectable marker is designed to determine whether the insertion of the transformant screening the transformed cells with a vector, that is, the target nucleic acid molecule, drug resistance, nutritional requirement, given the selectable phenotype such as expression of the resistance or the surface protein on cytotoxic agent markers that can be used. Since the selective agent in a process environment (selective agent) survive only cells expressing a selectable marker, or reflect a different phenotype, it may be selected for transformed cells.

[45]

[46]

As one more aspect of the present application, the present application also provides a microorganism producing 5'-inosinate which comprises a vector of the protein variant, a polynucleotide or present application coding for a protein variant of the application of the present application. More specifically, the microorganism includes a polynucleotide encoding the protein variant and / or the protein variants may be a microorganism which is produced by the transformed with a vector comprising a polynucleotide encoding a mutant protein is not limited thereto.

[47]

[48]

The term "transgenic" in this application is meant to allow the protein to the polynucleotide encoding the expression by introducing a vector comprising a polynucleotide encoding the target protein in the host cell in a host cell. If the transformed polynucleotide can be as long as expression in a host cell, is inserted in the chromosome of the host cell may be located, or include both of these positions in addition to the chromosome, or no matter what. In addition, the polynucleotides include DNA and RNA encoding the target protein. The polynucleotide so long as it can be expressed is introduced into a host cell, it does not matter whether it is to be introduced in any form. For example, the polynucleotide may be introduced into a host cell in the form of there is expressed by itself in an expression cassette (cassette expression) gene construct containing all the elements required. The expression cassette may include a promoter that is normally operably linked to the polynucleotide (promoter), a transcription termination signal, ribosome binding site and translation termination signal. The expression cassette may be an expression vector form a self-replicable. In addition, the polynucleotide is introduced into a host cell in the form of itself, and may be, which is possibly connected with the operation sequence necessary for the expression in a host cell, and the like.

[49]

In addition, it means that the term "operably linked to" the promoter sequence and the gene rea sequence to initiate and mediate the transcription of the polynucleotide encoding the target protein of the present application in the above is operatively connected to.

[50]

[51]

As used in this application, "microorganism producing 5'-inosinate" refers to 5 & apos; to the parent strain does not naturally ability of a microorganism or a 5'-inosinate 5'-inosinate with producing ability producing ability and / or discharge means the ability of micro-organisms give inosinate and production capacity or emissions. Microorganism which produces the 5'-inosinate in this application may be used interchangeably with a microorganism having a 5'-inosinate microorganism or emptying to discharge the 5'-inosinate.

[52]

Microorganism producing the above is 5'-inosinate, including variant proteins, a polynucleotide encoding the protein variants for discharging the 5'-inosinate of the present application, or a vector comprising a polynucleotide encoding the protein variants as cells or microorganisms transformed is converted to express the mutant protein, the purpose of this application the host cell of the "micro-organisms to produce a 5'-inosinate" or microorganism capable of producing 5'-inosinate, including the variant proteins If microorganisms which can be both. For example, Escherichia ( Escherichia ) genus, Serratia marcescens ( Serratia ), An air Winiah ( Erwinia ) genus, Enterobacter bacteria ( Enterobacteria ) genus, Salmonella ( Salmonella ) genus Streptomyces ( Streptomyces genus), Pseudomonas ( Pseudomonas ) genus Brevibacterium ( Brevibacterium ) in or Corynebacterium ( Corynebacterium can contain microorganism strain, etc.), a may be a specifically Corynebacterium spp.

[53]

The term "genus Corynebacterium to produce 5'-inosinate (the genus in the description of Corynebacterium ) microorganism" refers to a genus Corynebacterium microorganisms that have 5'-inosinate production capacity through the wild-type or mutant . Specifically, enhanced by the present application 5'-inosinate genus Corynebacterium microorganism having a production capacity is, or to enhance the activity of the gene is inserted into or associated with the inherent weakness gene native strain itself or outside the production mechanism in a 5'-inosinate It refers to a genus Corynebacterium microorganisms have 5'-inosinate production capacity. More specifically to the genus Corynebacterium microorganism having a 5'-inosinate producing ability in the present application comprises a mutant protein or a polynucleotide encoding it for discharging the 5'-inosinate of the present application, or encoding the protein variants is transformed with a vector including a polynucleotide, refers to the genus Corynebacterium microorganism have enhanced 5'-inosinate producing ability. The "enhanced 5'-inosinate producing ability The microorganism of the genus Corynebacterium have 'is transformed pre-change means a 5'-inosinate enhanced microbial production ability than the parent strain or non-modified microorganism. The "non-modified microorganism, is a vector including a polynucleotide encoding a microorganism, or protein variants for discharging the 5'-inosinate which does not include the mutant protein for discharging the wild-type strain or itself, the 5'-inosinate It refers to a transgenic non-transformed microorganism.

[54]

In one embodiment of the present application, the microorganisms of the present application adenylate succinate synthase (adenylosuccinate synthetase) and / or IMP dehydrogenase (IMP dehydrogenase) is added to the genus Corynebacterium microorganism weakening in the activity of a one can.

[55]

"Corynebacterium spp" in the present application is specifically Corynebacterium glutamicum ( of Corynebacterium glutamicum ), Corynebacterium ammoniagenes's Ness ( of Corynebacterium ammoniagenes ), Brevibacterium lactose flops momentum ( Brevibacterium lactofermentum ) , Brevibacterium Plastic pan ( Brevibacterium flavu m), Corynebacterium thermo amino to Ness ( Corynebacterium thermoaminogenes ), Corynebacterium epi syeonseu ( Corynebacterium efficiens ), Corynebacterium stay Yorkshire varnish ( Corynebacterium stationis) or the like , it is not limited thereto.

[56]

[57]

As one more aspect of the present application, the 5'-inosinate provide a production method that includes culturing a microorganism of the genus Corynebacterium in a medium to produce the 5'-inosinate.

[58]

Specifically, the method of the present application may further comprise the step of recovering the 5'-inosinate in the microorganism or the medium.

[59]

In the method, the method comprising culturing the microorganism, it can be not specifically limited, known batch culture method, the continuous culture method, performed by a fed-batch culture method. At this time, the culture conditions, particularly for but not limited to, a basic compound to an appropriate pH using: (phosphoric acid or sulfuric acid for example) (for example, pH 5 to 9, in particular (for example, sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound may be adjusted to pH 6 to 8, most specifically at pH 6.8), oxygen or oxygen-containing gas mixture is introduced to the culture to maintain the aerobic conditions. The culture temperature is 20 to 45 ℃, specifically, can be maintained for 25 to 40 ℃, but can be cultured for about 10 to 160 hours, without being limited thereto. The 5'-inosinate produced by the culture can be secreted into the culture medium or remains in the cell.

[60]

In addition, the medium for the culture to be used per a carbon source and a carbohydrate (such as glucose, sucrose trehalose, lactose, fructose, maltose, know three, starch and cellulose), maintenance, and fat (such as soybean oil, sunflower seed oil, peanut oil and coconut oil), fatty acids (e.g. palmitic acid, stearic acid and linoleic acid), alcohols (for example, glycerol and ethanol) and organic acids (e.g. acetic acid) can be used by using individually or mixed, such as, but , but it is not limited thereto. The nitrogen source may include nitrogen-containing organic compounds (e.g., peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean bakbun and urea), or inorganic compounds (e.g., ammonium ammonium ammonium sulfate, chloride, phosphate, ammonium carbonate and ammonium nitrate), but it can be used by using individually or mixed, such as, but not limited thereto. Although the source of the phosphate can be individually used, or a mixture of monobasic potassium phosphate, potassium susoyi, such as the corresponding sodium-containing salts to, but is not limited thereto. In addition, the culture medium and other metal salts (such as magnesium sulfate or iron sulfate), essential amino acids, and growth, such as vitamins can include a promoting material.

[61]

The method for recovering the 5'-inosinate produced in the incubation step of the present application can be by using a suitable method known in the art according to the culture method to collect the desired 5'-inosinate from the culture broth. For example, this can be used centrifugation, filtration, anion exchange chromatography, crystallization and HPLC, etc., it can be recovered from the culture medium or the desired 5'-inosinate microorganism by using the appropriate method known in the art.

[62]

In addition, the recovery step may comprise a purification step, may be performed using a suitable method known in the art. Thus, the 5'-inosinate which number of the microorganism may be a fermentation broth containing the purified form or a 5'-inosinate.

[63]

[64]

As yet another aspect of the present application, provides for the production of 5'-inosinate composition comprising a polynucleotide of the present application 5'-inosinate exhaust protein or encoding the variants.

[65]

The compositions of the present application may include, without limitation, a structure capable of operating the polynucleotide further. In the composition of the present application, the polynucleotide may be of a type that is included in the vector able to express a gene operably linked to the introduced in a host cell.

[66]

In addition, the composition may further comprise any suitable excipients commonly used in compositions for the production of 5'-inosinate. Such excipients include, for example, preservatives, wetting agents, dispersing agents, suspending agents, or the like, but a buffer, a stabilizer or isotonic agent, and the like.

[67]

[68]

This application is yet another one embodiment, provides a 5'-inosinate, for the production of increased production, the genus Corynebacterium microorganism of the protein of the present application.

[69]

The present application provides yet another one embodiment, the 5'-inosinate exhaust protein mutant Corynebacterium spp method, an increase in the 5'-inosinate emissions comprising enhanced in.

[70]

The term "5'-inosinate exhaust protein", "enhanced" and "microbial genus Corynebacterium" is as described above.

[71]

This application is yet another one embodiment, provides, for the production of a protein of the present application, the genus Corynebacterium 5'-inosinate emissions increase in the microorganism.

[72]

Mode for the Invention

[73]

[74]

It is described in more detail by the following Examples in the present application. However, these examples are for explaining the present application by way of example and, not necessarily the scope of the present application limited by these embodiments, it will be apparent to those skilled in the art of the present application.

[75]

[76]

Example 1: Excavation IMP exhaust protein

[77]

[78]

For the Corynebacterium involved in the IMP emissions to just identified a protein Corynebacterium Nice Stay Yorkshire ( of Corynebacterium stationis was manufactured's genomic DNA library of) ATCC6872. Since then, the genus Corynebacterium wild strains do not produce the IMP, or even produce IMP since very only be a very small amount is produced, in order to verify the IMP producing ability, producing CJI0323 strain ATCC6872 comes with IMP producing ability It was. Using ATCC6872 to genomic DNA library of the strain was carried out making CJI0323 screen for a membrane protein involved in the IMP discharge. Specific experiments are as follows.

[79]

[80]

Example 1-1: IMP production strains Screening Day CJI0323

[81]

ATCC6872 in order to produce a state of producing the IMP ATCC6782-derived in a phosphate buffer (pH7.0) or citrate buffer (pH5.5) 10 7 ~ 10 8 are suspended in cell / ml. Here to the UV treatment causes a mutation. Washed with 0.85% sodium chloride solution twice, and by diluting the material to confer resistance on a minimal medium containing 1.7% agar in a culture medium containing an appropriate concentration to give a smear after colony. Culturing individual colonies in a nutrient medium and was cultured for 24 hours in a seed medium. 3-4 days incubation resulting in a fermentation medium, the product IMP accumulated in the culture were selected for the best colony. In order to produce a high concentration of IMP production primary requirement adenine, guanine leakage type, lysozyme sensitivity, 3,4-dihydro-proline-resistant, streptomycin resistant, azetidine carboxylic acid-resistant, tear-resistant proline, serine aza-resistant, sulfamic guanidine resistance, Nord valine resistance, tri meto was carried out the above process in order to impart resistance in sequence for each material Supreme, the tolerance for the material was given and the final selection excellent CJI0323 IMP producing ability. In Table 1 are shown by comparing the degree of resistance against CJI0323 ATCC6872.

[82]

[83]

TABLE 1
characteristic ATCC6872 CJI0323
Adenine requirement Biyo configuration Requirement
Guanine leakage type Biyo configuration Leak-type
Lysozyme sensitivity 80 ug / ml 8 ug / ml
3,4-dihydro-proline-resistant 1000 ug / ml 3500 ug / ml
Streptomycin resistant 500 ug / ml 2000 ug / ml
Azetidine carboxylic acid tolerance 5 mg/ml 30 mg/ml
Thiazol-proline-resistant 10 ug / ml 100 ug / ml
Aza-serine-resistant 25 ug / ml 100 ug / ml
Guanidine preached tolerance 50 ug / ml 200 ug / ml
Norvaline resistance 0.2 mg/ml 2 mg/ml
Mezzo-resistant tree Supreme 20 ug / ml 100 ug / ml

[84]

[85]

- minimal medium: Glucose 2%, sodium 0.3%, phosphoric acid of claim 1, potassium 0.1%, the second 0.3% potassium phosphate, 0.3% magnesium sulfate, calcium chloride 10mg / l, ferrous sulfate 10mg / l, zinc sulfate 1mg / l, manganese chloride 3.6mg / l, L- cysteine ​​20mg / l, calcium pantothenate 10mg / l, thiamine hydrochloride 5mg / l, biotin, 30ug / l, adenine 20mg / l, guanine 20mg / l, pH7.3

[86]

[87]

- nutrient medium: peptone 1%, meat 1%, 0.25% sodium chloride, yeast extract, 1%, 2% agar, pH 7.2

[88]

[89]

- seed medium: glucose 1%, peptone 1%, meat 1%, 1% yeast extract, 0.25% sodium chloride, adenine 100mg / l, guanine 100mg / l, pH 7.5

[90]

[91]

- a fermentation medium 0.1% sodium glutamate, 1% ammonium chloride, 1.2% magnesium sulfate, 0.01% calcium chloride, ferrous sulfate 20mg / l, manganese sulfate 20mg / l, zinc sulfate 20mg / l, copper sulfate 5mg / l, L- Cysteine ​​23mg / l, was added to be alanine 24mg / l, nicotinic acid 8mg / l, biotin 45㎍ / l, thiamine hydrochloride 5mg / l, adenine 30mg / l, phosphoric acid (85%) of 1.9%, 2.55% glucose, 1.45% of fructose by using .

[92]

[93]

Example 1-2: CJI0323 entry into force of the potency test

[94]

Species after the diameter of the medium dispensed in 2ml 18mm test tube and pressure sterilization, respectively inoculated and cultured for 24 hours with shaking at 30 ℃ temperature and CJI0323 ATCC6872 was used as a seed culture. After dispensing the fermentation broth 29ml in 250ml Erlenmeyer flask for shaking and sterilized 15 minutes at 121 ℃ pressurized temperature, and inoculated with the seed culture 2ml were cultured for 3 days. The culture conditions were controlled as a rotation speed 170rpm, temperature 30 ℃, pH 7.5.

[95]

After the completion of the culture was measured by the method for production of IMP using HPLC (LC20A SHIMAZDU), culture results are shown in Table 2 below.

[96]

[97]

TABLE 2
Strain name IMP (g/L)
ATCC6872 0
CJI0323 9.52

[98]

[99]

The CJI0323 strain and Corynebacterium by naming a Nice Stay Yorkshire CN01-0323 deposit date of 11 July 2017 on international deposit under the Budapest Treaty on microorganisms organization Conservation Center Korea (Korean CultureCenter of Microorganisms, KCCM) given an accession number KCCM12151P received.

[100]

[101]

Example 1-3: Excavating emissions protein

[102]

It was added for further IMP in the minimum by the addition of a 1.7% agar medium was established screening criteria showing the growth decrease (growth inhibition) of CJI0323 strain. ATCC6872 to transform the genomic library plasmid with electroporation to CJI0323 strains and (van der Rest et al. 1999), were selected colonies are grown in a reduced release the added medium conditions excess IMP. The nucleotide sequence from the sequencing technique to obtain the plasmid from selected colonies was analyzed. This was identified from a membrane protein 1 species involved sikineunde release the reduced growth in excess of the added IMP conditions.

[103]

Corynebacterium Solarium of said one type of membrane protein is the amino acid sequence of SEQ ID NO: 2, and SEQ ID NO: 4 nucleotide sequence: was identified to be (NCBI GenBank NZ_CP014279, WP_066795121, MFS transporter). The film was known as MFS transporter protein, but did not confirm a definite function, and further features of the IMP emissions is not known. In the present application it was named as ImpE2 (WT).

[104]

[105]

Example 2: ImpE1, ImpE2 Identification

[106]

[107]

Example 2-1: impE1, impE2 OK

[108]

The film was confirmed that the gene construct of SEQ ID NO: 4 in the NCBI to find out the functions of the protein ImpE2 (NCBI GenBank: NZ_CP014279, WP_066795121 , MFS transporter). impE2 ( SEQ ID NO 4) ORF start of 7bp other gene located in the upstream thereof: it was confirmed that overlapping (NCBI GenBank NZ_CP014279, WP_066795119, transcriptional regulator) and 7bp. impE2 the protein encoded from the gene and the gene is located on the upstream is still did not resolve the function, in the application it was named as ImpE1 (WT) (amino acid sequence and the base sequence of SEQ ID NO: 3, SEQ ID NO: 1).

[109]

[110]

Example 2-2: impE1 or impE2 deficient vector production

[111]

If carried sikyeoteul defect in Examples 1 and IMP-producing strain or the ImpE1 ImpE2 that involved in releasing a growth reduction due to the sympathetic IMP through 2-1, to produce a defect vector for each gene in order to ensure that reduced emission capability IMP It was.

[112]

Gene segments to produce a vector was obtained via a PCR ATCC6872 to the genomic DNA as a template.

[113]

Specifically, the impE1 PCR for the SEQ ID NOS: 5,6 and primers SEQ ID NO: 7, 8 primers, impE2 PCR was performed using SEQ ID NOS: 9 and 10 primers and SEQ ID NO: 11, a primer 12 (Table 3).

[114]

[115]

TABLE 3
SEQ ID NO: primer Sequence (5'-3 ')
5 impE1 cup 1 GCTCTAGACGAGAAAGCTAAAGCCGGTGA
6 impE1 cup-2 GTTTTTAGCTACCATTGTTACACCCCGTGCAAGTTT
7 impE1 cup-3 GCACGGGGTGTAACAATGGTAGCTAAAAACTCCACC
8 impE1 cup-4 GCTCTAGAAATAGTTGGGGAAGTCCACTC
9 impE2 header-1 GCTCTAGACTTGGATGACCTGGTGGAAAA
10 impE2 header-2 CTTGGAGAAAATTTCCTACCATTCCAGTCCTTTCGT
11 impE2 header-3 GGACTGGAATGGTAGGAAATTTTCTCCAAGGGAAAT
12 impE2 header-4 GGACTAGTGGATTGTGTTGACGCACGATG
13 impE1E2kop-2 CTTGGAGAAAATTTCTGTTACACCCCGTGCAAGTTT
14 impE1E2kop-3 GCACGGGGTGTAACAGAAATTTTCTCCAAGGGAAAT

[116]

[117]

At this time, using primer Corynebacterium stationis (ATCC6872) gene (s) in the National Institutes of Health Gene Bank (NIH GenBank): was prepared on the basis of information on the (NCBI Genbank NZ_CP014279) and the surrounding nucleotide sequence.

[118]

Conditions of the PCR method was carried out for 5 min denaturation at 94 ℃, and then repeat 25 1 minutes 72 ℃ 94 ℃ 30 cho denaturation, 52 ℃ 3 bun annealing, polymerization time, polymerization at 72 ℃ 5 minutes of reaction. Subjected to SEQ ID NO: 5 and Primer 6, SEQ ID NO: 7 and nested polymerase chain reaction, the amplified gene impE1 two fragments using the primer as a template to 8 were obtained polynucleotide template of 1.8 kbp. A fragment of the obtained gene was digested with a restriction enzyme XbaI. T4 ligase pDZ of the line was cut using a kinase to XbaI restriction enzyme to the gene fragment (the Republic of Korea Patent No. 10-0924065 and International Publication Patent No. 2008-033001), and the vector was prepared pDZ- △ impE1. In addition, SEQ ID NO: 9 and 10 primers with the amplified impE2 gene fragment and SEQ ID NO: 11 and amplified using the primers 12 impE2 was subjected to nested PCR with the two gene fragments as the template to obtain a template polynucleotide of the 1.7kbp . A fragment of the obtained gene as a XbaI restriction enzyme was cut out, speI. The T4 ligase was cloned into pDZ vector of the line cut by the kinase to the XbaI restriction enzyme to the gene fragment, to prepare a pDZ- △ impE2.

[119]

[120]

Example 2-3: impE1, impE2 integrated deficient vector production

[121]

A gene encoding a protein involved in the release of growth reduction due to the IMP impE1 and impE2 because two superimposed gene may need to be adjusted at the same time. Thus impE1 and impE2 were all to produce a defective vector.

[122]

impE1 and impE2 PCR was performed with primers SEQ ID NO: 5 and 13 and SEQ ID NO: 14 and 12 primers. At this time, using primer Corynebacterium stationis (ATCC6872) gene (s) in the National Institutes of Health Gene Bank (NIH GenBank): was prepared on the basis of information on the (NCBI Genbank NZ_CP014279) and the surrounding nucleotide sequence. SEQ ID NO: 5 and 13, the primer with the amplified impE1 gene fragment and SEQ ID NO: 14 and amplified using the primers 12 impE2 subjected to nested PCR with the two gene fragments as the template and was obtained by the polynucleotide template of 2.0kbp. A fragment of the obtained gene was digested with XbaI, respectively, speI. The T4 ligase was cloned into pDZ vector of the line cut by the kinase to the XbaI restriction enzyme to the gene fragment, to prepare a pDZ- △ impE1E2.

[123]

[124]

Example 2-4: impE1, impE2 deficient production strain

[125]

Carried out after the transformation into the two kinds as in the electroporation method 1 kinds of plasmids prepared in Example 2-3 to each CJI0323 prepared in Example 2-2 (Appl Microbiol.Biotechnol (1999) 52:.. 541- 545 transfection method used), the strain by the recombinant vector is inserted into a chromosome of a homologous sequence according to was screened in medium containing kanamycin (kanamycin) 25 mg / L. The selected primary isolates was further carried out secondary cross (cross-over). Gene defect if the final transformed strains was confirmed by performing PCR using SEQ ID NO: 5, 8, and SEQ ID NO: 9, 12, and SEQ ID NO: 5 and 12 primer pairs.

[126]

The selected strain is CJI0323_ △ impE1, named CJI0323_ impE2 △, △ CJI0323_ impE1E2 and to evaluate the production performance of the IMP of the strain.

[127]

[128]

Species to the diameter of the medium dispensed in 2ml 18mm test tube, and sterilized after pressing, CJI0323, CJI0323_ △ impE1, CJI0323_ △ impE2, CJI0323_ △ inoculated impE1E2 24 hours with shaking at 30 ℃ temperature culture was used as seed culture. After dispensing the fermentation broth 29ml in 250ml Erlenmeyer flask for shaking and sterilized 15 minutes at 121 ℃ pressurized temperature, and inoculated with the seed culture 2ml were cultured for 3 days. The culture conditions were controlled as a rotation speed 170rpm, temperature 30 ℃, pH 7.5.

[129]

By a method using HPLC After the completion of the culture was measured production of IMP, culture results are shown in Table 4 below.

[130]

[131]

TABLE 4
Strain name IMP (g/L)
CJI0323 9.52
CJI0323_ △ impE1 1.92
CJI0323_ △ impE2 1.88
CJI0323_ △ impE1E2 1.80

[132]

[133]

At this time, the parent strain of Corynebacterium stay Yorkshire varnish CJI0323 the result of a comparison within the IMP accumulation medium, prepared under CJI0323_ △ impE1, CJI0323_ △ impE2, CJI0323_ △ the same conditions impE1E2 strains as shown in Table 4 of parent strain confirmed that the concentration decreases about 8g / L of IMP and was identified as ImpE1, ImpE2 the proteins involved in the IMP discharge.

[134]

[135]

Example 3: Production of IMP state CJI0323 impE1, impE2 determine the nucleotide sequence

[136]

[137]

For CJI0323 strains to high concentration IMP produced in the above Example 1, there is a possibility that improves performance IMP discharge to produce a high concentration of IMP. Therefore, the strain CJI0323 impE1, impE2 was undertaken to determine whether the variation of. Polymerization with the chromosomal DNA of CJI0323 chain reaction method was amplified by (the "PCR method" referred to). Specifically, first, in two minutes Taq DNA polymerase from using the SEQ ID NO: 15 and 16 primers, as a template the chromosomal DNA of the CJI0323 (Table 5) and 30 seconds bond and 1 min denaturation, 58 ℃ at 94 ℃, 72 ℃ the conditions of polymerizing fragments of about 2.8kb base pairs was amplified by PCR method to repeat 28 times.

[138]

[139]

Table 5
SEQ ID NO: primer Sequence (5'-3 ')
15 impE1E2 seqF GAACGGAGTCATCTCCTTTGC
16 impE1E2 seqr CCAAACGCTCTGCAAGAAACTG

[140]

[141]

Analysis of the nucleotide sequence it the same primers results, compared to the nucleotide sequence of the wild-type ATCC6872, impE1 was confirmed that the 490th nucleotide in g of the gene is substituted with a. This means that the variation of the 164th amino acid, glutamic acid ImpE1 protein is substituted with lysine.

[142]

Also, impE2 the fourth nucleotide of the gene g is replaced with a (mean that the 666th nucleotide in g of impE1 gene substituted with a), the 191st nucleotides of g was found that is substituted with a. This means that the second amino acid is replaced with the isoleucine of valine (corresponding to the 222 th amino acids of the protein ImpE1), the 64th amino acid of the protein is substituted at glycine ImpE2 as glutamic acid.

[143]

impE1 nucleotides CJI0323 strain impE1_CJI0323 (SEQ ID NO: 143), protein ImpE1_CJI0323 designated as (SEQ ID NO: 141) and, impE2 nucleotides CJI0323 strain impE2_CJI0323 (SEQ ID NO: 144), protein was named ImpE2_CJI0323 (SEQ ID NO: 142).

[144]

[145]

Example 4: impE1, impE2 mutation restored

[146]

[147]

Example 4-1: impE1 or impE2 variation vector restore production

[148]

In the IMP producing strain CJI0323 strain in Example 3 impE1, impE2 result confirming whether a variation of impE1 1 to more, impE2 was confirmed that contains two mutations on. CJI0323 strains because strain producing the IMP with a high concentration is likely to be variations of the mutation improves the IMP emptying. Therefore, after restoration of a wild type with no mutation ImpE of natural, for each mutant to ensure that give it the IMP emptying was carried out the following experiments.

[149]

To produce a vector restoring the wild type strain of Corynebacterium Natural Stay Yorkshire varnish ( Corynebacterium stationis ) it was performed by a PCR the ATCC6872 as a template. Amplified using primers SEQ ID NO: 17 and 18 impE1impE2 was treated with a restriction enzyme, a gene fragment of a XbaI, and cloned into the XbaI restriction enzyme located in the pDZ vector to prepare a pDZ-impE1E2 (WT).

[150]

[151]

Example 4-2: impE1 or impE2 alone disparity vector production

[152]

The natural wild-type strain of Corynebacterium stay Yorkshire varnish to produce a vector of the E164K mutation alone ImpE1 (Corynebacterium stationis) to the ATCC6872 as a template was used to SEQ ID NO: 19 and 20 primers and SEQ ID NO: 21 and 22 primers. Of SEQ ID NO: 19 and 1.8 by implementing the E164K-1 gene fragment and SEQ ID NO: 21 and amplified using 22 primer E164K-2 overlap the gene two fragments as a template, polymerase chain reaction kbp amplified using 20 primer a polynucleotide template It could be obtained. A fragment of the obtained gene was digested with a restriction enzyme XbaI. The T4 ligase was cloned into pDZ vector of the line cut by the kinase to the XbaI restriction enzyme to the gene fragment, to prepare a pDZ-impE1 (E164K).

[153]

To the ATCC6872 as a template to produce a vector of ImpE2 V2I single mutation it was used to SEQ ID NO: 19 and 23 primers and SEQ ID NO: 24 and 22 primers. Of SEQ ID NO: 19 and a 1-V2I 1.8kbp subjected to gene fragment and SEQ ID NO: 24 and 22 using the primer amplified 2-V2I superimposing the two gene fragments as a template, PCR amplification using the primer 23 to a polynucleotide template It could be obtained. A fragment of the obtained gene was digested with a restriction enzyme XbaI. The T4 ligase was cloned into pDZ vector of the line cut by the kinase to the XbaI restriction enzyme to the gene fragment, to prepare a pDZ-impE2 (V2I).

[154]

To the ATCC6872 as a template to produce a vector of the G64E mutation alone ImpE2 was used to SEQ ID NO: 19 and 25 primers and SEQ ID NO: 26 and 22 primers. Of SEQ ID NO: 19 and the 1.8kbp subjected to overlapping PCR with primers 25, a G64E-1 gene fragment and SEQ ID NO: 26 and a G64E-2 gene amplification with the two primer 22 fragment amplified using as a template a polynucleotide template It could be obtained. A fragment of the obtained gene was digested with a restriction enzyme XbaI. The T4 ligase was cloned into pDZ vector of the line cut by the kinase to the XbaI restriction enzyme to the gene fragment, to prepare a pDZ-impE2 (G64E).

[155]

[156]

TABLE 6
SEQ ID NO: primer Sequence (5'-3 ')
17 impE1E2 WT F GCTCTAGAGAACGGAGTCATCTCCTTTGC
18 impE1E2 WT R GCTCTAGACCAAACGCTCTGCAAGAAACTG
19 impE1 164K-1 GCTCTAGACTTGGATGACCTGGTGGAAAA
20 impE1 164K-2 CTGGGGCGCGTTGTTTTTCAGGATGCTCCCGAAGACG
21 impE1 164K-3 AACAACGCGCCCCAGAATTGG
22 impE1 164K-4 GCTCTAGAAATAGTTGGGGAAGTCCACTC
23 impE2 V2I 2 TGGAGTTTTTAGCTATCATTCCAGTCCTTTCGTGTAA
24 impE2 V2I 3 TAGCTAAAAACTCCACCCCAA
25 impE2 G64E-2 CCGAAAATCATCTGCTCCAAAGAGCTCATCAGCATGG
26 impE2 G64E-3 GCAGATGATTTTCGGTTCCGC

[157]

[158]

Example 4-3: impE1, impE2 mutation restoration and production single mutant

[159]

Carried out after the transformation into electroporation method the plasmid prepared in Example 4-1 in CJI0323 (Appl Microbiol.Biotechnol (1999) 52:.. Transfection method by using 541-545), the recombination of homologous sequences the strain vector is inserted into the chromosome were selected by the in medium containing kanamycin (kanamycin) 25 mg / L. The selected primary isolates was further carried out secondary cross (cross-over). Whether mutation restoration of the final transformed strains was confirmed by performing PCR using SEQ ID NO: 15 and 16 primers and sequenced. Since the production strain was named as CJI0323_impE1E2 (WT).

[160]

CJI0323_impE1E2 (WT) after switching exemplary transfection the three kinds of plasmids prepared in Example 4-2, the strain (Appl Microbiol.Biotechnol (1999) 52:.. 541-545 transformed by the conversion method used), recombination of homologous sequences the strain vector is inserted into the chromosome by screening was in medium containing kanamycin (kanamycin) 25 mg / L. The selected primary isolates was further carried out secondary cross (cross-over). Whether the final transfected gene mutation introduction of the transition strain was confirmed by performing PCR using SEQ ID NO: 15 and 16 primers and sequenced. The selected strain was named CJI0323_impE1 (E164K), CJI0323_impE2 (V2I), CJI0323_impE2 (G64E).

[161]

The Corynebacterium Stay Yorkshire Nice CJI0323_impE1 (E164K), Corynebacterium Stay Yorkshire Nice CJI0323_impE2 (V2I) and Corynebacterium Stay Yorkshire Nice CJI0323_impE2 (G64E) isolates the international deposit agency Conservation Center Korea microorganisms under the Budapest Treaty (Korean accession to 2 dated 11 January 2018, the Culture Center of Microorganisms, KCCM) and were each given an accession number KCCM12359P, KCCM12360P and KCCM12361P.

[162]

[163]

Example 4-4: impE1, impE2 integrated mutant strain produced

[164]

Example 4-2 The pDZ-impE2 (V2I), pDZ-impE2 (G64E) was transformed with the plasmid electroporation method to CJI0323_impE1 (E164K) (Appl Microbiol.Biotechnol produced in (1999) 52: 541 transfection method used), the strain by the recombinant vector is inserted into a chromosome of a homologous sequence according to -545 was selected on medium containing kanamycin (kanamycin) 25 mg / L. The selected primary isolates was further carried out secondary cross (cross-over). Whether the final transfected gene mutation introduction of the transition strain was confirmed by performing PCR using SEQ ID NO: 15 and 16 primers and sequenced. After the production strain was named CJI0323_impE1 (E164K) _impE2 (V2I), CJI0323_impE1 (164K) _impE2 (G64E).

[165]

pDZ-impE2 (G64E) the strain vector is inserted into the chromosome by recombination of homologous sequences was transformed by electroporation method and plasmid in CJI0323_impE2 (V2I) have medium containing kanamycin (kanamycin) 25 mg / L It was screened. The selected primary isolates was further carried out secondary cross (cross-over). Whether the final transfected gene mutation introduction of the transition strain was confirmed by performing PCR using SEQ ID NO: 15 and 16 primers and sequenced. The selected strain was named CJI0323_impE2 (V2I) (G64E).

[166]

[167]

Example 4-5: impE1, impE2 mutant strain evaluation

[168]

After the seed medium 2ml diameter dispensed into 18mm test tube and pressure sterilization, CJI0323_impE1E2 (WT), CJI0323_impE1 (E164K), CJI0323_impE2 (V2I), CJI0323_impE2 (G64E), CJI0323_impE1 (E164K) _impE2 (V2I), CJI0323_impE1 (E164K) _impE2 ( G64E), was inoculated CJI0323_impE2 (V2I) (G64E) for 24 hours with shaking at 30 ℃ temperature culture was used as seed culture. After dispensing the fermentation broth 29ml in 250ml Erlenmeyer flask for shaking and sterilized 15 minutes at 121 ℃ pressurized temperature, and inoculated with the seed culture 2ml were cultured for 3 days. The culture conditions were controlled as a rotation speed 170rpm, temperature 30 ℃, pH 7.5.

[169]

We measured the production of IMP by the method using HPLC After the completion of the culture, culture results are shown in the following table 7.

[170]

[171]

Table 7
Strain name IMP (g/L)
CJI0323 9.52
CJI0323_impE1E2(WT) 2.32
CJI0323_impE1(E164K) 2.57
CJI0323_impE2 (V2I) 3.11
CJI0323_impE2(G64E) 3.27
CJI0323_impE1(E164K)_impE2(V2I) 4.24
CJI0323_impE1(E164K)_impE2(G64E) 6.27
CJI0323_ impE2(V2I)(G64E) 7.35

[172]

[173]

For each mutation position as shown in the result, at least variation of two or three kinds of consolidation and integration mutation was confirmed that all involved in the IMP discharge. Therefore, even if the production does not produce the IMP in the genus Corynebacterium microorganisms that produce only infinitesimal, IMP increased production due to ImpE protein variants of the present application can be interpreted as very meaningful.

[174]

[175]

Example 5: impE1, substitution of an amino acid by another amino acid mutations impE2

[176]

[177]

Example 5-1: impE1, impE2 vector for making amino acid substitution mutations inserted

[178]

To via the 5'-inosinate results confirm the importance of the positions of the three kinds of typical variations (impE1 (E164K), impE2 (V2I), impE2 (G64E)) of the capability of production enhanced mutation, amino acid 164 in the amino acid sequence of impE1 It was produced in the mutagenic vector for replacing the second, or the 64th amino acid in the amino acid sequence of impE2 with other amino acids.

[179]

Vector construction process is as follows for the first ImpE1 (E164K) mutation introduced.

[180]

Based on the reported polynucleotide sequence Corynebacterium stay Yorkshire varnish to remove the chromosomal gene of CJI0323, by this, using the primer pair of SEQ ID NO: as the template 27 and each of SEQ ID NO: 28 ~ 45 gene through a polymerase chain reaction to give a fragment. Conditions of the PCR method was carried out for 5 min denaturation at 94 ℃, after repeated bun 72 ℃ 94 ℃ 30 cho denaturation, 55 ℃ 30 cho annealing, polymerization 20 times, in 72 ℃ polymerization 5 min. As a result, it can obtain a polynucleotide of the 18 kinds of 0.7kbp. Next Corynebacterium separated chromosomal gene of the stay Yorkshire CJI0323 varnish, using the primer pair SEQ ID NO: 46, respectively of SEQ ID NO: 47-64, respectively, a gene fragment was obtained by polymerase chain reaction. Conditions of the PCR method was carried out for 5 min denaturation at 94 ℃, after repeated bun 72 ℃ 94 ℃ 30 cho denaturation, 55 ℃ 30 cho annealing, polymerization 20 times, in 72 ℃ polymerization 5 min. As a result, it can obtain a polynucleotide of the 18 kinds of 0.7kbp.

[181]

The two fragments obtained through the results subjected to overlap extension PCR method as a template were obtained polynucleotide template of 18 kinds of 1.4kbp. Was cut to a fragment of the obtained gene with a restriction enzyme SpeI with T4 ligase and transformed into, connect the pDZ vector of the line was used to azepin digested with XbaI restriction enzyme in the gene fragment of E. coli DH5α containing the kanamycin (25mg / L) It was plated on the LB solid medium.

[182]

The primers used for the vector construction sequence information is given in Table 8.

[183]

[184]

Table 8
SEQ ID NO: primer Sequence (5'-3 ')
27 Spe1-impE1 164 1F GGGACTAGTGATTCCGGCCAACTGTCG
28　 impE1 164-R 1R TGGGGCGCGTTGGCGTTCAGGATGCTC
29　 impE1164-H 1R TGGGGCGCGTTGGTGTTCAGGATGCTC
30 impE1 164-D 1R TGGGGCGCGTTGATCTTCAGGATGCTC
31　 impE1 164-S 1R TGGGGCGCGTTGGGATTCAGGATGCTC
32　 impE1 164-T 1R TGGGGCGCGTTGGGTTTCAGGATGCTC
33　 impE1 164-N 1R TGGGGCGCGTTGGTTTTCAGGATGCTC
34 impE1 164-Q 1R TGGGGCGCGTTGCTGTTCAGGATGCTC
35　 impE1 164-C 1R TGGGGCGCGTTGGCATTCAGGATGCTC
36 impE1 164-G 1R TGGGGCGCGTTGGCCTTCAGGATGCTC
37 impE1 164-P 1R TGGGGCGCGTTGCGGTTCAGGATGCTC
38　 impE1 164-A 1R TGGGGCGCGTTGGGCTTCAGGATGCTC
39　 impE1 164-V 1R TGGGGCGCGTTGGACTTCAGGATGCTC
40　 impE1 164-I 1R TGGGGCGCGTTGGATTTCAGGATGCTC
41　 impE1 164-1R L TGGGGCGCGTTGCAGTTCAGGATGCTC
42　 impE1 164-M 1R TGGGGCGCGTTGCATTTCAGGATGCTC
43　 impE1 164-F 1R TGGGGCGCGTTGGAATTCAGGATGCTC
44　 impE1 164-Y 1R TGGGGCGCGTTGGTATTCAGGATGCTC
45　 In impE1 164-1R TGGGGCGCGTTGCCATTCAGGATGCTC
46　 Spe1-impE1 164 2R GGGACTAGTCATGAACTTGCCGCGCTC
47　 impE1 164-R 2F GAGCATCCTGAACGCCAACGCGCCCCA
48 impE1 164-H 2F GAGCATCCTGAACACCAACGCGCCCCA
49　 impE1 164-D 2F GAGCATCCTGAAGATCAACGCGCCCCA
50　 impE1 164-S 2F GAGCATCCTGAATCCCAACGCGCCCCA
51　 impE1 164-T 2F GAGCATCCTGAAACCCAACGCGCCCCA
52　 impE1 164-N 2F GAGCATCCTGAAAACCAACGCGCCCCA
53　 impE1 164-Q 2F GAGCATCCTGAACAGCAACGCGCCCCA
54　 impE1 164-C 2F GAGCATCCTGAATGCCAACGCGCCCCA
55　 impE1 164-G 2F GAGCATCCTGAAGGCCAACGCGCCCCA
56 impE1 164-P 2F GAGCATCCTGAACCGCAACGCGCCCCA
57　 impE1 164-A 2F GAGCATCCTGAAGCCCAACGCGCCCCA
58 impE1 164-V 2F GAGCATCCTGAAGTCCAACGCGCCCCA
59　 impE1 164-I 2F GAGCATCCTGAAATCCAACGCGCCCCA
60　 impE1 164 L 2F GAGCATCCTGAACTGCAACGCGCCCCA
61　 impE1 164-M 2F GAGCATCCTGAAATGCAACGCGCCCCA
62　 impE1 164-F 2F GAGCATCCTGAATTCCAACGCGCCCCA
63　 impE1 164-Y 2F GAGCATCCTGAATACCAACGCGCCCCA
64 impE1 164-W 2F GAGCATCCTGAATGGCAACGCGCCCCA

[185]

[186]

After the screening of colonies transformed with a vector conversion gene is inserted object by PCR using a plasmid extraction method known commonly obtained was obtained a plasmid Plasmid information are given in Table 9.

[187]

[188]

Table 9
number Plasmid people
1 PDZ-164R impE1
2 PDZ-164H impE1
3 PDZ-164D impE1
4 PDZ-164S impE1
5 PDZ-164T impE1
6 PDZ-164N impE1
7 PDZ-164Q impE1
8 PDZ-164C impE1
9 PDZ-impE1 164G
10 PDZ-164P impE1
11 PDZ-164A impE1
12 PDZ-164V impE1
13 PDZ-164I impE1
14 PDZ-164L impE1
15 PDZ-impE1 164m
16 PDZ-164F impE1
17 PDZ-impE1 164Y
18 PDZ-impE1 164W

[189]

[190]

Second ImpE2 (V2I) manufacturing process for the disparity vector is introduced as follows.

[191]

Based on the reported polynucleotide sequence Corynebacterium stay Yorkshire varnish to remove the chromosomal gene of CJI0323, by this, using the primer pair of SEQ ID NO: 65 as a template and each of SEQ ID NOS: 66-83 gene by polymerase chain reaction to give a fragment. Conditions of the PCR method was carried out for 5 min denaturation at 94 ℃, after repeated bun 72 ℃ 94 ℃ 30 cho denaturation, 55 ℃ 30 cho annealing, polymerization 20 times, in 72 ℃ polymerization 5 min. As a result, it can obtain a polynucleotide of the 18 kinds of 0.7kbp. Next Corynebacterium separated chromosomal gene of the stay Yorkshire CJI0323 varnish, using a primer pair of SEQ ID NO: 84, respectively of SEQ ID NO: 85-102, respectively, a gene fragment was obtained by polymerase chain reaction. Conditions of the PCR method was carried out for 5 min denaturation at 94 ℃, after repeated bun 72 ℃ 94 ℃ 30 cho denaturation, 55 ℃ 30 cho annealing, polymerization 20 times, in 72 ℃ polymerization 5 min. As a result, it can obtain a polynucleotide of the 18 kinds of 0.7kbp.

[192]

The two fragments obtained through the results subjected to overlap extension PCR method as a template were obtained polynucleotide template of 18 kinds of 1.4kbp. Was cut to a fragment of the obtained gene with a restriction enzyme XbaI with T4 ligase and transformed into, connect the pDZ vector of the line was used to azepin digested with XbaI restriction enzyme in the gene fragment of E. coli DH5α containing the kanamycin (25mg / L) It was plated on the LB solid medium. The primer sequence information are shown in Table 10, used for the vector construction.

[193]

[194]

[Table 10]
SEQ ID NO: Primers people Sequence (5'-3 ')
65 XbaI-impE2 2 1F GGGTCTAGATTGCATGCTGTGCAAGA
66　 impE2 2-R 1R GGAGTTTTTAGCGCGCATTCCAGTCCT
67　 impE2 2-H 1R GGAGTTTTTAGCGTGCATTCCAGTCCT
68　 impE2 2-K 1R GGAGTTTTTAGCCTTCATTCCAGTCCT
69　 impE2 2-D 1R GGAGTTTTTAGCGTCCATTCCAGTCCT
70　 impE2 2-E 1R GGAGTTTTTAGCTTCCATTCCAGTCCT
71　 impE2 2-S 1R GGAGTTTTTAGCGGACATTCCAGTCCT
72　 impE2 2-T 1R GGAGTTTTTAGCGGTCATTCCAGTCCT
73 impE2 2-N 1R GGAGTTTTTAGCGTTCATTCCAGTCCT
74　 impE2 2-Q 1R GGAGTTTTTAGCCTGCATTCCAGTCCT
75　 impE2 2-C 1R GGAGTTTTTAGCGCACATTCCAGTCCT
76 impE2 2-G 1R GGAGTTTTTAGCGCCCATTCCAGTCCT
77　 impE2 2-P 1R GGAGTTTTTAGCTGGCATTCCAGTCCT
78　 impE2 2-A 1R GGAGTTTTTAGCAGCCATTCCAGTCCT
79　 impE2 2-L 1R GGAGTTTTTAGCCAGCATTCCAGTCCT
80　 impE2 2-M 1R GGAGTTTTTAGCCATCATTCCAGTCCT
81　 impE2 2-F 1R GGAGTTTTTAGCGAACATTCCAGTCCT
82　 impE2 2-Y 1R GGAGTTTTTAGCGTACATTCCAGTCCT
83　 impE2 2-W 1R GGAGTTTTTAGCCCACATTCCAGTCCT
84　 XbaI-impE2 2 2R GGGTCTAGATTGCTCGCCCACGCGCA
85 impE2 2-R 2F AGGACTGGAATGCGCGCTAAAAACTCC
86　 impE2 2-H 2F AGGACTGGAATGCACGCTAAAAACTCC
87　 impE2 2-K 2F AGGACTGGAATGAAGGCTAAAAACTCC
88　 impE2 2-D 2F AGGACTGGAATGGACGCTAAAAACTCC
89　 impE2 2-E 2F AGGACTGGAATGGAAGCTAAAAACTCC
90　 impE2 2-S 2F AGGACTGGAATGTCCGCTAAAAACTCC
91　 impE2 2-T 2F AGGACTGGAATGACCGCTAAAAACTCC
92　 impE2 2-N 2F AGGACTGGAATGAACGCTAAAAACTCC
93　 impE2 2-Q 2F AGGACTGGAATGCAGGCTAAAAACTCC
94　 impE2 2-C 2F AGGACTGGAATGTGCGCTAAAAACTCC
95　 impE2 2-G 2F AGGACTGGAATGGGCGCTAAAAACTCC
96　 impE2 2-P 2F AGGACTGGAATGCCAGCTAAAAACTCC
97　 impE2 2-A 2F AGGACTGGAATGGCTGCTAAAAACTCC
98　 impE2 2-L 2F AGGACTGGAATGCTGGCTAAAAACTCC
99　 impE2 2-M 2F AGGACTGGAATGATGGCTAAAAACTCC
100　 impE2 2F 2F AGGACTGGAATGTTCGCTAAAAACTCC
101　 impE2 2-Y 2F AGGACTGGAATGTACGCTAAAAACTCC
102　 impE2 2-W 2F AGGACTGGAATGTGGGCTAAAAACTCC

[195]

[196]

After the screening of colonies transformed with a vector conversion gene is inserted object by PCR using a plasmid extraction method known commonly obtained was obtained a plasmid Plasmid information are given in Table 11.

[197]

[198]

[Table 11]
number Plasmid people
1 PDZ-2R impE2
2 PDZ-2H impE2
3 PDZ-impE2 2K
4 PDZ-2D impE2
5 PDZ-2E impE2
6 PDZ-2S impE2
7 PDZ-2T impE2
8 pDZ-impE2 2N
9 PDZ-impE2 2Q
10 PDZ-2C impE2
11 PDZ-impE2 2G
12 PDZ-2P impE2
13 PDZ-impE2 2A
14 PDZ-2L impE2
15 pDZ-impE2 2M
16 pDZ-impE2 2F
17 PDZ-2Y impE2
18 pDZ-impE2 2W

[199]

[200]

Vector construction process is as follows for the last ImpE2 (G64E) mutagenic.

[201]

Based on the reported polynucleotide sequence Corynebacterium stay Yorkshire varnish to remove the chromosomal gene of CJI0323, by this, using the primer pair as a template SEQ ID NO: 103, respectively of SEQ ID NO: 104-121 gene via polymerase chain reaction to give a fragment. Conditions of the PCR method was carried out for 5 min denaturation at 94 ℃, after repeated bun 72 ℃ 94 ℃ 30 cho denaturation, 55 ℃ 30 cho annealing, polymerization 20 times, in 72 ℃ polymerization 5 min. As a result, it can obtain a polynucleotide of the 18 kinds of 1kbp. Next Corynebacterium separated chromosomal gene of the stay Yorkshire CJI0323 varnish, using a primer pair of SEQ ID NO: 122, respectively of SEQ ID NO: 123-140 respectively, to obtain a gene fragment by the polymerase chain reaction. Conditions of the PCR method was carried out for 5 min denaturation at 94 ℃, after repeated bun 72 ℃ 94 ℃ 30 cho denaturation, 55 ℃ 30 cho annealing, polymerization 20 times, in 72 ℃ polymerization 5 min. As a result, it can obtain a polynucleotide of the 18 kinds of 1kbp.

[202]

The two fragments obtained through the results subjected to overlap extension PCR method as a template were obtained 18 kinds of the template polynucleotide 2kbp. Was cut to a fragment of the obtained gene with a restriction enzyme XbaI with T4 ligase and transformed into, connect the pDZ vector of the line was used to azepin digested with XbaI restriction enzyme in the gene fragment of E. coli DH5α containing the kanamycin (25mg / L) It was plated on the LB solid medium.

[203]

The primers used for the vector construction is the same as the sequence information table 12.

[204]

[205]

[Table 12]
SEQ ID NO: primer Sequence (5'-3 ')
103 XbaI-impE2 64 1F GGGTCTAGAAAAGAGCTTAAGGCAGCTGCT
104　 impE2 64-R 1R GAAAATCATCTGGCGCAAAGAGCTCAT
105　 impE2 64-H 1R GAAAATCATCTGGTGCAAAGAGCTCAT
106　 impE2 64-D 1R GAAAATCATCTGGTCCAAAGAGCTCAT
107　 impE2 64-K 1R GAAAATCATCTGCTTCAAAGAGCTCAT
108　 impE2 64-S 1R GAAAATCATCTGGGACAAAGAGCTCAT
109　 impE2 64-T 1R GAAAATCATCTGGGTCAAAGAGCTCAT
110　 impE2 64-N 1R GAAAATCATCTGGTTCAAAGAGCTCAT
111　 impE2 64-Q 1R GAAAATCATCTGCTGCAAAGAGCTCAT
112　 impE2 64-C 1R GAAAATCATCTGGCACAAAGAGCTCAT
113　 impE2 64-P 1R GAAAATCATCTGTGGCAAAGAGCTCAT
114　 impE2 64-A 1R GAAAATCATCTGAGCCAAAGAGCTCAT
115　 impE2 64-V 1R GAAAATCATCTGGACCAAAGAGCTCAT
116　 impE2 64-I 1R GAAAATCATCTGGATCAAAGAGCTCAT
117　 impE2 64-L 1R GAAAATCATCTGCAGCAAAGAGCTCAT
118　 impE2 64-M 1R GAAAATCATCTGCATCAAAGAGCTCAT
119　 impE2 64-F 1R GAAAATCATCTGGAACAAAGAGCTCAT
120　 impE2 64-Y 1R GAAAATCATCTGGTACAAAGAGCTCAT
121　 impE2 64-W 1R GAAAATCATCTGCCACAAAGAGCTCAT
122　 XbaI-impE2 64 2R GGGTCTAGACGGTCAATGAAGTCTCAACGG
123 impE2 64-R 2F ATGAGCTCTTTGCGCCAGATGATTTTC
124 impE2 64-H 2F ATGAGCTCTTTGCACCAGATGATTTTC
125 impE2 64-D 2F ATGAGCTCTTTGGACCAGATGATTTTC
126 impE2 64-K 2F ATGAGCTCTTTGAAGCAGATGATTTTC
127 impE2 64-S 2F ATGAGCTCTTTGTCCCAGATGATTTTC
128 impE2 64-T 2F ATGAGCTCTTTGACCCAGATGATTTTC
129 impE2 64-N 2F ATGAGCTCTTTGAACCAGATGATTTTC
130 impE2 64-Q 2F ATGAGCTCTTTGCAGCAGATGATTTTC
131 impE2 64-C 2F ATGAGCTCTTTGTGCCAGATGATTTTC
132 impE2 64-P 2F ATGAGCTCTTTGCCACAGATGATTTTC
133 impE2 64-A 2F ATGAGCTCTTTGGCTCAGATGATTTTC
134 impE2 64-V 2F ATGAGCTCTTTGGTCCAGATGATTTTC
135 impE2 64-I 2F ATGAGCTCTTTGATCCAGATGATTTTC
136 impE2 64-L 2F ATGAGCTCTTTGCTGCAGATGATTTTC
137 impE2 64-M 2F ATGAGCTCTTTGATGCAGATGATTTTC
138 impE2 64-F 2F ATGAGCTCTTTGTTCCAGATGATTTTC
139 impE2 64-Y 2F ATGAGCTCTTTGTACCAGATGATTTTC
140 impE2 64-W 2F ATGAGCTCTTTGTGGCAGATGATTTTC

[206]

[207]

After the screening of colonies transformed with a vector conversion gene is inserted object by PCR using a plasmid extraction method known commonly obtained was obtained a plasmid Plasmid information are given in Table 13.

[208]

[209]

[Table 13]
number Plasmid people
1 PDZ-64R impE2
2 PDZ-64H impE2
3 PDZ-64D impE2
4 PDZ-impE2 64K
5 PDZ-64S impE2
6 PDZ-64T impE2
7 PDZ-64N impE2
8 PDZ-64Q impE2
9 PDZ-64C impE2
10 PDZ-64P impE2
11 PDZ-64A impE2
12 PDZ-64V impE2
13 PDZ-64I impE2
14 PDZ-64L impE2
15 PDZ-impE2 64M
16 PDZ-64F impE2
17 PDZ-impE2 64Y
18 PDZ-64W impE2

[210]

[211]

Example 5-2: ImpE1, the amino acid position of the mutation in the mutant strain produced ImpE2 5'-inosinate and substituted with other amino acid-producing ability compared

[212]

Example 5-1 to a side of 54 kinds of vectors for introduction prepared in the transformed Corynebacterium stay Yorkshire varnish CJI0323 and the strain by the recombinant vector is inserted into a chromosome of a homologous sequence is kanamycin (kanamycin) 25 mg / L were selected in a medium containing. The selected primary isolates was further carried out secondary cross (cross-over). Whether the final transfected gene mutation introduction of the transition strain was confirmed by performing PCR using SEQ ID NO: 15 and 16 primers and sequenced. Strain due to the inserted mutant of them as shown in the following Table 14.

[213]

[214]

[Table 14]
number Strain name
1 CJI0323::impE1(E164R)
2 CJI0323::impE1(E164H)
3 CJI0323::impE1(E164D)
4 CJI0323::impE1(E164S)
5 CJI0323::impE1(E164T)
6 CJI0323::impE1(E164N)
7 CJI0323::impE1(E164Q)
8 CJI0323::impE1(E164C)
9 CJI0323::impE1(E164G)
10 CJI0323::impE1(E164P)
11 CJI0323::impE1(E164A)
12 CJI0323::impE1(E164V)
13 CJI0323::impE1(E164I)
14 CJI0323::impE1(E164L)
15 CJI0323::impE1(E164M)
16 CJI0323::impE1(E164F)
17 CJI0323 :: impE1 (E164Y)
18 CJI0323::impE1(E164W)
19 CJI0323::impE2(V2R)
20 CJI0323 :: impE2 (V2H)
21 CJI0323::impE2(V2K)
22 CJI0323::impE2(V2D)
23 CJI0323 :: impE2 (V2E)
24 CJI0323::impE2(V2S)
25 CJI0323 :: impE2 (V2T)
26 CJI0323::impE2(V2N)
27 CJI0323 :: impE2 (V2Q)
28 CJI0323::impE2(V2C)
29 CJI0323::impE2(V2G)
30 CJI0323::impE2(V2P)
31 CJI0323 :: impE2 (V2A)
32 CJI0323 :: impE2 (V2L)
33 CJI0323 :: impE2 (V2M)
34 CJI0323::impE2(V2F)
35 CJI0323 :: impE2 (V2Y)
36 CJI0323::impE2(V2W)
37 CJI0323::impE2(G64R)
38 CJI0323::impE2(G64H)
39 CJI0323:impE2(G64D)
40 CJI0323::impE2(G64K)
41 CJI0323::impE2(G64S)
42 CJI0323::impE2(G64T)
43 CJI0323::impE2(G64N)
44 CJI0323::impE2(G64Q)
45 CJI0323::impE2(G64C)
46 CJI0323::impE2(G64P)
47 CJI0323::impE2(G64A)
48 CJI0323::impE2(G64V)
49 CJI0323::impE2(G64I)
50 CJI0323:impE2(G64L)
51 CJI0323::impE2(G64M)
52 CJI0323::impE2(G64F)
53 CJI0323:impE2(G64Y)
54 CJI0323::impE2(G64W)

[215]

[216]

Example 1 and cultured in the same way, were analyzed for the concentration of 5'-inosinate therefrom (Table 15).

[217]

[218]

[Table 15]
impE1, impE2 mutant combinations introducing primary 5'-inosinate production level (g / L)
Strain Average 5'-inosinate
Controls CJI0323_impE1E2(WT) 2.32
1 CJI0323::impE1(E164R) 9.42
2 CJI0323::impE1(E164H) 8.47
3 CJI0323::impE1(E164D) 7.37
4 CJI0323::impE1(E164S) 8.56
5 CJI0323::impE1(E164T) 8.85
6 CJI0323::impE1(E164N) 9.13
7 CJI0323::impE1(E164Q) 7.45
8 CJI0323::impE1(E164C) 7.37
9 CJI0323::impE1(E164G) 9.13
10 CJI0323::impE1(E164P) 9.43
11 CJI0323::impE1(E164A) 7.44
12 CJI0323::impE1(E164V) 8.18
13 CJI0323::impE1(E164I) 8.09
14 CJI0323::impE1(E164L) 7.85
15 CJI0323::impE1(E164M) 7.39
16 CJI0323::impE1(E164F) 7.56
17 CJI0323 :: impE1 (E164Y) 7.60
18 CJI0323::impE1(E164W) 8.56
19 CJI0323::impE2(V2R) 7.99
20 CJI0323 :: impE2 (V2H) 8.75
21 CJI0323::impE2(V2K) 8.66
22 CJI0323::impE2(V2D) 8.28
23 CJI0323 :: impE2 (V2E) 9.32
24 CJI0323::impE2(V2S) 6.37
25 CJI0323 :: impE2 (V2T) 8.37
26 CJI0323::impE2(V2N) 9.80
27 CJI0323 :: impE2 (V2Q) 7.04
28 CJI0323::impE2(V2C) 7.23
29 CJI0323::impE2(V2G) 7.71
30 CJI0323::impE2(V2P) 7.80
31 CJI0323 :: impE2 (V2A) 6.57
32 CJI0323 :: impE2 (V2L) 6.42
33 CJI0323 :: impE2 (V2M) 9.20
34 CJI0323::impE2(V2F) 9.43
35 CJI0323 :: impE2 (V2Y) 8.37
36 CJI0323::impE2(V2W) 7.22
37 CJI0323::impE2(G64R) 4.42
38 CJI0323::impE2(G64H) 5.14
39 CJI0323::impE2(G64D) 11.53
40 CJI0323::impE2(G64K) 4.8
41 CJI0323::impE2(G64S) 5.7
42 CJI0323::impE2(G64T) 5.52
43 CJI0323::impE2(G64N) 5.9
44 CJI0323::impE2(G64Q) 4.8
45 CJI0323::impE2(G64C) 5.9
46 CJI0323::impE2(G64P) 4.75
47 CJI0323::impE2(G64A) 4.58
48 CJI0323::impE2(G64V) 4.56
49 CJI0323::impE2(G64I) 5.89
50 CJI0323::impE2(G64L) 5.6
51 CJI0323::impE2(G64M) 4.3
52 CJI0323::impE2(G64F) 5.89
53 CJI0323::impE2(G64Y) 4.6
54 CJI0323::impE2(G64W) 4.76

[219]

[220]

As the result, all of the mutant strain has been increased IMP producing ability compared to respective control, the variation location of the three species may be confirmed that the relevant location variations affecting the IMP emptying increase in ImpE protein.

[221]

[222]

Example 6: IMP production share-based impE1, impE2 mutagenic

[223]

[224]

Example 6-1: IMP production share-based impE1, impE2 mutant strain produced

[225]

The activity of succinate synthase (adenylosuccinate synthetase) and IMP dehydrogenase (IMP dehydrogenase) in ATCC6872 as adenylate for the degradation pathways of the IMP to determine the mutagenic effect of impE1, impE2 weakening in the IMP-producing strain the strain was produced. The first start codon by changing the second nucleotide from a to t encoding the two enzymes in the nucleotide sequence of each gene purA and guaB was changed. The ATCC6872 in which the expression of the two genes is attenuated strain was named CJI9088. Produced in Example 4-2 in the manufacture CJI9088 strain pDZ-impE1 (E164K), pDZ-impE2 (V2I), pDZ-impE2 transform (G64E) by electroporation method, and CJI9088_impE1 (E164K) _impE2 ( the embodiment of pDZ- impE2 (G64D) vector produced in example 5-1 to V2I) was transformed by electroporation method. Strains by the recombinant vector is inserted into a chromosome of a homologous sequence was selected in medium containing kanamycin 25mg / l. The selected primary isolates was carried out a second cross again. Whether the final transformants transgenic for the conversion strain was confirmed by performing PCR using SEQ ID NO: 15 and 16, the primer pair.

[226]

The fabricated CJI9088 and CJI9088_impE1 (E164K), were evaluated CJI9088_impE2 (V2I), CJI9088_impE2 (G64E) and CJI9088_impE1 (E164K) _impE2 (V2I) (G64D) the strain of IMP producing ability. We measured the production of IMP by the method using HPLC After the completion of the culture, culture results are shown in the following table 16.

[227]

[228]

[Table 16]
Strain name IMP (g/L)
CJI9088 0.52
CJI9088_impE1(E164K) 0.84
CJI9088_impE2(V2I) 0.93
CJI9088_impE2(G64E) 1.73
CJI9088_impE1(E164K)_impE2(V2I)(G64D) 4.30

[229]

[230]

Confirming the accumulation of IMP resulting culture medium, it was confirmed that the increase of the parental strain CJI9088 IMP producing ability compared to more than 61% up to 727%. Therefore, IMP increased production due to ImpE protein variants of the present application can be interpreted as very meaningful.

[231]

[232]

From the above description, those skilled in the filed will appreciate that this application without changing the technical spirit or essential features may be embodied in other specific forms. In this regard, the embodiments described above are only to be understood as exemplary rather than limiting in all aspects. The scope of the present application should be construed as the meaning and scope, and all such modifications as derived from the equivalent concepts of the following claims rather than the foregoing description within the scope of the present application.

[233]

[234]

[235]

[236]

[237]

Claims

[Claim 1]

In SEQ ID NO: 1, 164 amino acid, the mutant protein in which one or more amino acids selected from the second amino acid, and SEQ ID NO: 2, the group consisting of the 64th amino acid in SEQ ID NO: 2 discharging the 5'-inosinate substituted with other amino acids.

[Claim 2]

The method of claim 1, wherein the 164 amino acid protein is a variant of the discharge, 5'-inosinate substituted with lysine, arginine, asparagine, glycine, threonine or proline, in the SEQ ID NO: 1.

[Claim 3]

The method of claim 1, wherein the second amino acid is a protein variant of the discharge, the 5'-inosinate substituted with isoleucine, phenylalanine, methionine, glutamic acid, histidine, or asparagine in the SEQ ID NO: 2.

[Claim 4]

The method of claim 1, wherein the protein variant to 64th amino acids from the SEQ ID NO: 2 is discharged, the 5'-inosinate substituted by aspartate, glutamate, asparagine, cysteine, isoleucine, or phenylalanine.

[Claim 5]

The polynucleotide encoding the mutant protein of claim 1.

[Claim 6]

Claim 5 vector containing the polynucleotide.

[Claim 7]

Mutant protein of claim 1, claim 5 poly genus Corynebacterium microorganism producing 5'-inosinate which comprises a nucleotide or claim 6 vector.

[Claim 8]

The method of claim 7, wherein the microorganism of the genus Corynebacterium is Corynebacterium stay Yorkshire varnish ( Corynebacterium stationis ) of 5'-inosinate genus Corynebacterium microorganism to produce.

[Claim 9]

The method of claim 7, wherein the microorganism adenylate a succinate synthase (adenylosuccinate synthetase) or IMP dehydrogenase (IMP dehydrogenase) Corynebacterium which is active to produce the added, 5'-inosinate weakening in the Solarium spp.

[Claim 10]

Claim 7, comprising the step of culturing in a medium a microorganism of Corynebacterium, 5'-inosinate method.

[Claim 11]

11. The method of claim 10, the method comprising the steps of: 5'-inosinate method further comprising the step of recovering the 5'-inosinate in the microorganism or the medium.

[Claim 12]

11. The method of claim 10, wherein the microorganism of the genus Corynebacterium is Corynebacterium stay Yorkshire varnish ( Corynebacterium stationis a, 5'-inosinate method).

[Claim 13]

First, the genus Corynebacterium 5'-inosinate for the purpose of increasing the production of microbial protein in protest.

[Claim 14]

The mutant protein of claim 1 Corynebacterium method in increased emissions of Solarium, 5'-inosinate which comprises enhanced in the microorganism.

[Claim 15]

First, the genus Corynebacterium 5'-inosinate for the purpose of increasing the emissions of microbial protein in protest.